CN106636319B - Molecular biological method for rapid identification of eastern white-browed gibbon and northern white-cheeked gibbon - Google Patents

Abstract

The invention discloses a molecular biology method for rapidly identifying the eastern white-browed gibbon and the northern white-cheeked gibbon, belonging to the field of molecular biology. Separate and extract genomic DNA from the object to be tested as a template; perform PCR with P1 primer pair and P2 primer pair respectively to obtain fragments A and B; fragments A and B are spliced and sheared to obtain the complete COI gene sequence; Species identification by nucleotide sequence characteristic variation sites of Eastern white-browed gibbon and northern white-cheeked gibbon. The invention realizes the rapid and accurate molecular identification of the eastern white-browed gibbon and the northern white-cheeked gibbon for the first time, and makes up for many deficiencies of the traditional morphological identification method. The identification objects include venous blood, feces, hair or urine samples of animals, which are accurate, rapid and convenient, and play an important role and significance in maintaining ecosystems and protecting biodiversity.

Description

Molecular biological method for rapid identification of eastern white-browed gibbon and northern white-cheeked gibbon

technical field

The invention belongs to the field of molecular biology, and in particular relates to a molecular biology method that can quickly identify the Eastern White-browed Gibbon and the Northern White-cheeked Gibbon.

Background technique

There are 4 genera and 17 species in the family Hylobatidae. There are 3 genera and 6 species in China, namely Hoolock leuconedys, Eastern hoolock gibbon and Nomascus. leucogenys), western black crested gibbon (N. concolor), eastern black crested gibbon (N. nasutus), Hainan gibbon (N. hainanus) and the white-handed gibbon (Hylobates lar) in the genus Hylobates.

Both the Eastern White-browed Gibbon and the Northern White-cheeked Gibbon are distributed in Yunnan Province, China. The former is found in Dehong Prefecture and Baoshan City; the latter is found in Mengla and Luchun Huanglian Mountains in Xishuangbanna. In recent years, due to the shrinking and changing habitats and frequent poaching and hunting, the two species of gibbons are on a downward trend, and both have been listed as national first-level key protected wildlife and endangered by the International Union for Conservation of Nature (IUCN). .

In border trade animal smuggling and consumption, wild animals are mainly used for food, medicine, ornamental entertainment, or are made into handicrafts and decorations. However, the relevant departments such as entry-exit animal quarantine and forest public security are faced with a serious problem: how to carry out the rapid and accurate identification of these rare and endangered wild animals, broken and scattered tissues, organs and incomplete individuals, as well as processed products? The reality is that in many cases, due to the difficulty of material identification, the criminals cannot be punished in time. It was learned from the Yunnan Provincial Public Security Frontier Defense Corps that the Eastern White-browed Gibbon and the Northern White-cheeked Gibbon are both distributed in Yunnan and are often smuggled together with other wild apes. The incomplete morphological characteristics of scattered tissues and organs (such as skulls) or processed fur, such as bleaching and dyeing, often lead to inaccurate identification, which seriously affects the timeliness and effectiveness of case handling. Therefore, how to quickly and accurately identify two rare and endangered gibbons not only provides strong technical support for relevant departments such as entry-exit animal quarantine and forest public security, but also provides effective auxiliary support for the research and protection of the two species of gibbons.

Traditional methods of identification of gibbons mainly rely on their morphological characteristics. Taking the eastern white-browed gibbon as an example, it is dioecious, males are brown-black or dark brown, and adult males have two clearly separated white eyebrows; females are mostly gray or gray-yellow, with lighter eyebrows. The northern white-cheeked gibbon, on the other hand, has a slender body and is black in males, except that each cheek has a large white spot, and the tufted crested hair on the top is more pointed and longer. The female dark brown crown spot is triangular in shape. Most of the body is dirty yellow, and the dark brown of the chest and abdomen is rare.

Although the traditional morphological identification is clear and intuitive, it has major limitations: (1) the survival status of rare and endangered species is very fragile and the number is extremely limited; (2) it is difficult to realize the identification of broken and scattered tissues, organs and incomplete individuals and processed products. Identification; (3) The identification of rare and endangered animals can only rely on experienced classification experts, but the reality is that there are fewer and fewer classification experts. With the development of biodiversity protection, the protection of rare and endangered animals is gradually strengthened, and a new identification method is urgently needed to make up for the shortcomings of traditional methods.

In the 1990s, with the rapid development of molecular biology, especially the improvement of PCR technology, more and more molecular biology techniques were applied to species identification and classification.

Among them, the most commonly used molecular marker is the sequence of mitochondrial cytochrome C oxidase subunit I (Cytochrome Coxidase I, COI). It can detect subtle differences between species and individuals of the same species, and provides a new method for the classification and identification of closely related species and subspecies. It is not limited by incomplete materials and developmental stages, and makes up for the traditional morphology. Due to the many shortcomings of identification, it has become the first choice for species identification.

At present, only the complete mitochondrial genome sequence of northern white-cheeked gibbon (NC_021957) and the cytb sequence of western white-browed gibbon (Hoolock hoolock, western hoolock gibbon) (Y13304, 1141bp in length) are in Genbank. At home and abroad, there is no report on the use of COI gene fragments as molecular markers to distinguish between the Eastern White-browed Gibbon and the Northern White-cheeked Gibbon.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a molecular biology method capable of quickly and accurately distinguishing and identifying the eastern white-browed gibbon and the northern white-cheeked gibbon in view of the deficiencies of the prior art.

The purpose of the present invention is achieved through the following technical solutions.

A molecular biology method for rapidly identifying Eastern white-browed gibbon and northern white-cheeked gibbon, comprising the steps of the following sequence:

(1) Separating and extracting genomic DNA from the object to be tested;

(2) Use two sets of PCR primers to carry out PCR amplification of the full sequence of the COI gene of the object to be tested:

The primer sequences of the two groups of PCR primers are:

P1F: 5'-AACCGAACGCAAATCA-3',

P1R: 5'-AAAGTGTAGCCCGAGA-3';

P2F: 5'-TCCGCTGGCAGGAAACT-3',

P2R: 5'-GGCGTGGTCGTGGAAAG-3';

Taking the genomic DNA obtained in step (1) as a template, taking the described P1F and P1R as primer pairs to carry out PCR to obtain the fragment A PCR product; using the described P2F and P2R as primer pairs to carry out PCR to obtain the fragment B PCR product;

(3) After the well-amplified fragment A PCR product and fragment B PCR product are sent to the biological company for sequencing, the fragment A and the fragment B are spliced, and the redundant bases before and after the full sequence of the COI gene are cut off, that is, the COI of the object to be tested is obtained. full gene sequence;

(4) Species identification is carried out according to the nucleotide sequence characteristic variation sites listed in the following table:

Characteristic Variation Sites of Nucleotide Sequences in Eastern White-browed Gibbons and Northern White-cheeked Gibbons

The sequence numbers of the sites in the table are based on the full sequence position of the COI gene; the letter "M" in the table is the abbreviation of the Eastern White-browed Gibbon; the letter "J" is the abbreviation of the Northern White-cheeked Gibbon; the relevant degenerate base code is R= A/G, Y=C/T.

The PCR reaction system described in step (2) is 50 μl, including 25 ng of template DNA, 1× PCR buffer, 2.5 mM Mgcl ₂ , 1 mM dNTP, 2 μg/μl BSA, 2 pM of forward and reverse primers, and 1 unit of TaqDNA polymerase The specific operation is as follows: add deionized water to adjust the final volume to 50 μl, and seal the system with paraffin oil; PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 1 minute ; After 35 cycles, a further 10 min extension at 72°C.

The test object includes venous blood, feces, hair or urine samples of animals.

Compared with the prior art, the present invention has the following advantages:

1. The present invention realizes the rapid and accurate identification of the two kinds of gibbons according to the nucleotide sequence characteristic variation sites of the Eastern White-browed Gibbon and the Northern White-cheeked Gibbon disclosed for the first time, and makes up for many deficiencies of traditional morphological identification methods.

2. The present invention can identify processed samples of Eastern white-browed gibbon and northern white-cheeked gibbon that cannot be identified, incomplete or partially degraded from traditional morphology, and has the characteristics of accuracy, speed and convenience, and can usually be completed in 2 days. . It can reduce and avoid problems such as difficulty in transporting wild animals, especially live ones, large funding requirements, and difficult preservation and management, and provide strong technical support for timely and effective identification. Ultimately, it will greatly improve the efficiency of entry-exit animal quarantine and forest public security related departments; it will help to improve the intensity and effect of cracking down on illegal and criminal activities of various wildlife resources; it will play an important role in maintaining ecosystems and protecting biodiversity. role and contribution.

3. At the same time, because the eastern white-browed gibbon and the northern white-cheeked gibbon are both national first-level key protected animals and IUCN endangered grade animals, the COI gene sequence characteristic variation site disclosed by the present invention can be non-destructive. The samples of Eastern White-browed Gibbon and Northern White-cheeked Gibbon obtained by non-invasive sampling provide a yardstick, which is especially important in habitat research and protection of wild endangered species.

Description of drawings

Figure 1. The electrophoretic pattern (partial pattern) of the PCR amplification results of the conventional universal primers tried in the experiments of the present invention.

Figure 2. Based on the complete mitochondrial genome sequence of white-handed gibbon (NC_002082), the positions of two sets of PCR primers designed by the present invention.

The full length of primer P1 (underlined) is 1503 bp, and this pair of primers contains 1214 bp of CO1 sequence. The forward primer is located at 289 bp before the start codon of the CO1 gene sequence; the reverse primer is located at 1198 bp of the CO1 gene sequence (P1R is inverse and complementary to the sequence shown in Figure 2).

The full length of primer P2 (marked by a wavy line) is 1375 bp, and this pair of primers contains 1153 bp of CO1 sequence. The forward primer is located at 389 bp of the CO1 gene sequence; the reverse primer is located at 205 bp after the CO1 gene sequence (P2R is inverse and complementary to the sequence shown in Figure 2).

Figure 3. The electrophoretic patterns of the PCR amplification results of two sets of primers of the present invention based on the eastern white-browed gibbon "Beibei" and "Guagua" and the northern white-cheeked gibbon "Lele" and "Weiwei".

The electrophoresis channels 1 to 4 in the figure are the amplification results of the primer P1; the electrophoresis channels 5 to 8 are the amplification results of the primer P2. The source of the samples in the electrophoresis channels 1 and 5 is "Beibei" (venous blood); the source of the samples in the electrophoresis channels 2 and 6 is "Guagua" (feces); the source of the samples in the electrophoresis channels 3 and 7 is the northern white cheek The gibbon "Lele" (venous blood); the source of the electrophoresis channel 4 and 8 samples is the northern white-cheeked gibbon "Wei" (feces).

Figure 4. The electrophoresis pattern of the PCR amplification results of the two sets of primers of the present invention based on the samples of the Eastern White-browed gibbon "Bei Bei" obtained by non-invasive sampling method (feces, hair and urine).

The electrophoresis channels 1 to 3 in the figure are the amplification results of the primer P1; the electrophoresis channels 4 to 6 are the amplification results of the primer P2. Electrophoresis channel 1 and 4 samples are from "Beibei" (feces); electrophoresis channels 2 and 5 are from "Beibei" (hair); electrophoresis channels 3 and 6 are from "Eastern white-browed gibbon" Bebe" (urine).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the drawings and the examples are not intended to limit the present invention.

Example 1

1. Collection and preservation of samples

There are four samples used in the experiment of the present invention, including two eastern white-browed gibbons, "Beibei" and "Guagua"; and two northern white-cheeked gibbons, "Lele" and "Weiwei". The details of the samples are shown in Table 1.

Table 1. Samples used in the experiments of the present invention.

1.1 Blood samples

The sources of the samples were the eastern white-browed gibbon "Beibei" and the northern white-cheeked gibbon "Lele", and fresh venous blood was collected. Eastern white-browed gibbon "Bei Bei", female, 9 years old, rescued by Yunnan Wildlife Rescue Center in Dehong Prefecture, Yunnan Province in 2015. The northern white-cheeked gibbon "Lele", female, 18 years old, was rescued by the Yunnan Wildlife Rescue Center in Xishuangbanna Prefecture, Yunnan Province in 2006. The morphological identification of the two gibbons was completed by experts from the Kunming Institute of Zoology, Chinese Academy of Sciences, and they are currently kept in Yunnan Wildlife Park.

Use a new anticoagulant blood collection tube to collect 1ml (ml) of fresh whole blood (venous blood) of eastern white-browed gibbon and northern white-cheeked gibbon, and put 300ul (microliter) into 1.5ml Eppendrof sterilized by high temperature and high pressure steam. In a centrifuge tube, it is stored in the freezer layer of the refrigerator (-20°C) for DNA extraction. The rest of the blood samples were stored in the ultra-low temperature refrigerator (-80°C) of the Animal Teaching and Research Office of Southwest Forestry University. It should be noted that there are already anticoagulant blood collection tubes on the market, so there is no need to add anticoagulant reagents such as heparin.

1.2 Fecal samples

The so-called non-invasive sampling method is to collect feces, urine, shed hair or feathers, egg shells, food left by animals without harming, capturing, touching or even seeing the animals themselves. A method of sampling for genetic analysis of different forms of analytical samples such as debris (containing oral exfoliated cells), scales, etc. With the development of biodiversity protection, the protection of rare and endangered animals has been gradually strengthened, and the non-invasive sampling method has been widely used in the field of animal conservation genetics research.

Non-invasive sampling of feces has the greatest potential value, as it is the simplest of all non-invasive sampling methods and has the least impact on wildlife.

The sample sources were the Eastern white-browed gibbon "Guagua" and the northern white-cheeked gibbon "Wei Wei", and fresh feces were collected. Eastern white-browed gibbon "Guagua", male, adult (age unknown), from Dehong Prefecture, kept in Yunnan Wildlife Park since 2011. Northern white-cheeked gibbon "Wei Wei", male, adult (age unknown), from Xishuangbanna Prefecture, kept in Yunnan Wildlife Park since 2010. The fresh feces of the Eastern white-browed gibbon "Guagua" and the northern white-cheeked gibbon "Wei" were collected from the surface of the mucous membrane-rich part (gloves and masks were worn to avoid human contamination of the samples), and stored with 2 times the volume of 100% ethanol.

1.3 Hair samples

The source of the sample is the eastern white-browed gibbon "Bebe". Pick up the shed hair with follicles, bring it back to the laboratory and store it at -80°C for future use.

Take 5 hairs, cut off the hair roots, wash with double distilled water and absolute ethanol respectively, add 0.5ml TEN, containing 2% SDS, 40mmol/L DTT, 15ul proteinase K, keep overnight at 37°C in a water bath, fully lysed . DNA was extracted by conventional phenolic extraction method.

1.4 Urine samples

The source of the sample is the eastern white-browed gibbon "Bebe". The day before sampling, the agricultural film was laid on the inside of the cage; the urine was collected the next day, brought back to the laboratory and stored at -80°C for future use.

2. Extraction of sample DNA

2.1 DNA extraction from blood samples

Refer to the standard phenol-chloroform method (the standard phenol/chloroform protocol) or directly use the DNA extraction kit (Shanghai Huashun Bioengineering Co., Ltd.) to extract. During the whole DNA extraction process, pay attention to gentle and gentle movements, especially in the step of DNA precipitation, do not shake violently, so as to prevent DNA from breaking into small fragments, resulting in the inability of DNA molecules to form flocculent precipitates, which will ultimately affect the quality and quality of extracted DNA. concentration.

2.2 Fecal DNA extraction

Refer to the method of Wang Yuefeng et al. (2006) and make some improvements. The specific steps are as follows:

(1) Thoroughly mix the fecal samples soaked in alcohol, and use a pipette to draw 1 mL of fine fecal pellets into a 2 mL Eppendrof centrifuge tube.

(2) Add 1.5 mL of absolute ethanol, mix well, centrifuge, and pour off the supernatant; repeat several times until the supernatant is colorless.

(3) Add 1.5 mL of high-purity water, mix well, centrifuge, and remove the supernatant; repeat several times until the supernatant is colorless.

(4) 400uL of digestion solution (10mmol/L Tris-HCl, pH8.0; 0.1mol/L EDTA, pH8.0; 5g/LSDS) was added in a 55°C water bath for 5h.

(5) Centrifuge at 10 000 r/min for 5 min after digestion, take the supernatant and inject it into a centrifuge tube with 0.6 g starch, shake well and then centrifuge.

(6) Take the supernatant into a new Eppendrof centrifuge tube, add 200uL CTAB buffer (100mmol/L Tris-HCl, pH 8, 1.4mol/L NaCl, 20mmol/L EDTA, 2% CTAB), and water bath at 70°C for 20min.

(7) Add an equal volume of phenol/chloroform-isoamyl alcohol for extraction for 10 min, centrifuge at 10 000 r/min, and suck the supernatant into a new centrifuge tube; repeat 2 to 3 times.

(8) Use the PCR product purification kit E.Z.N.A.Cycle pure Kit (USA, Omega Bio-Tek Company) to purify the extracted product, elute the purified product with 100uL ultrapure water, and store at -20°C.

2.3 Hair DNA extraction

Take 5 eastern white-browed gibbon hairs, cut off the hair roots, soak in double distilled water and absolute ethanol respectively, add 0.5 ml TEN, containing 2% SDS, 40 mmol/L DTT, 15 ul proteinase K, and keep overnight in a water bath at 37 °C. fully lysed. DNA was extracted by conventional phenolic extraction method.

2.4 Urine DNA extraction

Urine DNA extraction refers to the method of Chen Ronghua et al. (2005) chelex-100.

3. References to primers

In the early stage of the experiment, we intend to use the DNA barcode primers commonly used in relevant literature at home and abroad to amplify the DNA barcode gene sequences of the Eastern White-browed Gibbon and the Northern White-cheeked Gibbon. The primer sequences we tried are shown in Table 2.

Table 2. Primer sequences attempted in the experiments of the present invention.

When using the primers of Chen Cuiping (2012), in addition to using two pairs of primers for amplification, we also used mixed primer sequences to amplify samples that could not be successfully amplified by two pairs of primers. In addition to the PCR amplification conditions and PCR reaction systems provided in the literature, we also used the following PCR amplification conditions and PCR reaction systems for amplification.

①The PCR reaction system is 50μl, including about 25ng of template DNA, 1×PCR buffer, 2.5mM Mgcl ₂ , 1mM dNTP, 2μg/μl BSA, 2pM of forward and reverse primers, and 1 unit of TaqDNA polymerase. Deionized water was added to adjust to a final volume of 50 μl, and the system was capped with paraffin oil. Reactions were performed on a RoboCycler Gradient 40 (Stratagene) thermal cycler.

②The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 50/52/55°C for 1 minute, and extension at 72°C for 1 minute. After 35 cycles, 72°C was extended for another 10 minutes.

5ul PCR amplification products were taken and electrophoresed on a 1% agarose gel (130V voltage, electrophoresis for 35 min). Gelview stain. The gel imaging system detects and the electrophoresis pattern is shown in Figure 1. In Figure 1: Marker bands from top to bottom are 5K, 3K, 2K, 1K, 750bp, 500bp, 250bp, 100bp, of which the concentration of the 750bp band is about 20ng/ul, and the concentration of other bands is about 10ng/ul.

The number of electrophoresis holes in the gel image, primers used for PCR amplification and annealing temperature are shown in Table 3.

Table 3. Electropherogram in Figure 1 of the electrophoresis hole number, primers used for PCR amplification and annealing temperature.

Electrophoresis well number PCR amplification primers Annealing temperature 1 LepF+LepR 50 2 LCO1490+HCO2198 50 3 LepF1_t1+LepR1_t1 50 4 VF1d_t1+VR1d_t1 50 5 LepF1_t1+LepR1_t1+VF1d_t1+VR1d_t1 50 6 LepF+LepR 52 7 LCO1490+HCO2198 52 8 LepF1_t1+LepR1_t1 52 9 VF1d_t1+VR1d_t1 52 10 LepF1_t1+LepR1_t1+VF1d_t1+VR1d_t1 52 11 LepF+LepR 55 12 LCO1490+HCO2198 55

Unfortunately, after repeated adjustment of the PCR reaction system and PCR amplification conditions, the target amplification band could not be obtained.

4. Primer Design

Download the mitochondrial genome sequence of Hylobates lar, GenBank accession number.NC_002082 from the public database of NCBI (http://www.ncbi.nlm.nih.gov/), and manually design it using Primer5 software Primers, primers use Primer5 software to check primer mismatch, dimer and hairpin structure, and design two sets of primers by themselves. The primer sequences are:

The P1F forward primer is: 5'-AACCGAACGCAAATCA-3',

The P1R reverse primer is: 5'-AAAGTGTAGCCCGAGA-3';

The P2F forward primer is: 5'-TCCGCTGGCAGGAAACT-3',

The P2R reverse primer was: 5'-GGCGTGGTCGTGGAAAG-3'.

The specific positions of the two sets of primers are shown in FIG. 2 .

The full length of primer P1 (underlined) is 1503 bp, and this pair of primers contains 1214 bp of CO1 sequence. The forward primer is located at 289 bp before the start codon of the CO1 gene sequence; the reverse primer is located at 1198 bp of the CO1 gene sequence (P1R is inverse and complementary to the sequence shown in Figure 2).

The full length of primer P2 (marked by a wavy line) is 1375 bp, and this pair of primers contains 1153 bp of CO1 sequence. The forward primer is located at 389 bp of the CO1 gene sequence; the reverse primer is located at 205 bp after the CO1 gene sequence (P2R is inverse and complementary to the sequence shown in Figure 2).

5. PCR amplification reaction system and reaction conditions

①The PCR reaction system is 50μl, including about 25ng of template DNA, 1×PCR buffer, 2.5mM Mgcl ₂ , 1mM dNTP, 2μg/μl BSA, 2pM of forward and reverse primers, and 1 unit of TaqDNA polymerase. Deionized water was added to adjust to a final volume of 50 μl, and the system was capped with paraffin oil. Reactions were performed on a RoboCycler Gradient 40 (Stratagene) thermal cycler. The DNA concentration of the sample varies from 0.02ug/ml to 0.4ug/ml, and it is diluted with sterile deionized water.

②The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 1 minute. After 35 cycles, 72°C was extended for another 10 minutes. The annealing temperature varies from 50°C to 55°C.

5ul PCR amplification products were taken and electrophoresed on a 1% agarose gel (130V voltage, electrophoresis for 35 min). Gelview stain. The gel imaging system detects and the electrophoresis pattern is shown in Fig. 3 and Fig. 4 . In accompanying drawing 3 and accompanying drawing 4: Marker band is 5K, 3K, 2K, 1K, 750bp, 500bp, 250bp, 100bp sequentially from top to bottom, wherein 750bp band concentration is about 20ng/ul, and other band concentration is about 10ng /ul.

6. Gene Sequencing

Select 3-5 PCR amplification products with better amplification effect and send them to a biological company for purification and sequencing (the primers used for sequencing are the same as those used for PCR). The sequencing reaction was performed on an ABI 377 automatic sequencer (Applied Biosystems) using the BigDyeTerminator kit (V2.0) of Applied Company under the conditions suggested by the manufacturer. Both forward and reverse chains were measured.

PCR was performed by using P1F and P1R as primer pairs to obtain fragment A; PCR was performed by using P2F and P2R as primer pairs to obtain fragment B. After splicing Fragment A and Fragment B, perform sequence comparison with Clustal W software in MEGA software package with the corresponding sequence of white-handed gibbon (NC_002082) downloaded from the Internet, find the start codon ATG and stop codon, and truncate the entire COI gene sequence The redundant bases before and after are obtained to obtain the complete sequence of the COI gene of the sample to be tested. See Gene Sequence Listing for details.

7. Screening of sequences

Mitochondrial pseudogenes (Numts or mitochondrial pseudogenes) have been found in the target mitochondrial gene fragments obtained after amplification and sequencing in many vertebrates, especially in primates (Jayaprakash et al., 2015; Calvignac et al., 2011) . Therefore, after obtaining the data, the identification of the sequence is very important. The sequence we obtained has a strong preference for base composition, and base G only accounts for a small percentage (16.7-16.8%). In the second base composition of codons in the coding region, T or C accounted for a higher percentage (40% and 25.5-25.9%), and there was no stop codon during amino acid translation. Based on the above characteristics, it is indicated that the sequence we obtained is a mitochondrial gene fragment.

8. Species identification based on sequence feature variation sites

The electropherograms analyzed by the sequencer were combined with Seqman in DNASTAR to splicing forward and reverse strands. A total of four sequences are obtained in the experiment of the present invention, two sequences of the eastern white-browed gibbon, "Beibei" and "Guagua"; two sequences of the northern white-cheeked gibbon, "Lele" and "Weiwei".

After downloading the COI gene fragment corresponding to the complete mitochondrial genome sequence (NC_021957) of the northern white-cheeked gibbon, five sequences in total were analyzed together with the four sequences obtained in the experiment of the present invention. The five sequences were sorted with Clustal W, and the sequence variation analysis was performed with MEGA 2.1, resulting in Table 5.

Table 5. Characteristic variation sites of nucleotide sequences in Eastern white-browed gibbon and northern white-cheeked gibbon. The sequence number of the locus is based on the position of the complete sequence of the COI gene. The letter "M" in the table is the abbreviation of the Eastern White-browed Gibbon; the letter "J" is the abbreviation of the Northern White-cheeked Gibbon. The relevant degenerate bases codes are R=A/G, Y=C/T.

Table 5 shows the characteristic variation sites of nucleotide sequences in the Eastern white-browed gibbon and the northern white-cheeked gibbon. The position of the variant site is read from top to bottom. For example, the first variant site of the COI gene of the eastern white-browed gibbon and the northern white-cheeked gibbon is the nucleotide position 10, and the second variant position The point is the 42nd position of the nucleotide sequence, and so on, the last variation site is the 1541st position of the nucleotide sequence.

During the specific identification, the COI gene sequence of the object to be tested was directly compared with Table 5. We take 5 variant sites as an example to illustrate. At the variant sites 10, 42, 60, 61, and 66, we found that the nucleotide sequence of ATGTC is the Eastern White-browed Gibbon; GCACY is the Northern White-cheeked Gibbon, where Y can be either C or T, both are Northern white-cheeked gibbon. The same is true for other degenerate bases. If it is neither ATGTC nor GCACY to be tested, it indicates neither the Eastern White-browed Gibbon nor the Northern White-cheeked Gibbon. Based on these variant loci, we will process eastern white-browed gibbon and northern white-cheeked gibbon samples that are not identifiable by traditional morphology, mutilated or partially degraded.

Example 2

The object to be tested is a piece of muscle tissue with an area of about 0.3 × 0.4 cm and a weight of about 1.6 g. It was provided by the Yunnan Provincial Forest Public Security Bureau. It is suspected to be one of the eastern white-browed gibbon and the northern white-cheeked gibbon.

Take about 100 mg of the object to be tested in the sample tube, and cut the tissue block into pieces with clean scissors. Directly use DNA extraction kit (Shanghai Huashun Bioengineering Co., Ltd.) to extract. During the extraction process, it is necessary to add a little more RNase A to minimize RNA residues to ensure DNA quality.

The PCR amplification reaction system, PCR reaction conditions and gene sequence determination were all consistent with the samples in Example 1.

The COI gene sequence of the test object is directly compared with Table 5. For example, first looking at the position of nucleotide No. 10, the result shows that the base to be tested is "A"; secondly, looking at the position of nucleotide No. 42, the base to be tested is "T". By analogy, we can finally conclude that the source of the muscle tissue sample is the Eastern White-browed Gibbon.

Example 3

The object to be tested is a piece of fur, with an area of about 1.3 × 1.6 cm, provided by the Yunnan Provincial Forest Public Security Bureau. It is suspected to be one of the eastern white-browed gibbon and the northern white-cheeked gibbon.

Hair DNA extraction, PCR amplification reaction system, PCR reaction conditions and gene sequence determination are all consistent with the samples in Example 1.

The COI gene sequence of the test object is directly compared with Table 5. For example, first looking at the position of nucleotide No. 10, the result shows that the base to be tested is "G"; secondly, looking at the position of nucleotide No. 42, the base to be tested is "C". By analogy, we can finally conclude that the source of the fur tissue sample is the northern white-cheeked gibbon.

Example 4

In order to compare the 3 genera of gibbons that are commonly distributed in China, except for the complete COI gene sequences of the eastern white-browed gibbon (Gibbon genus) and the northern white-cheeked gibbon (Gibbon genus) disclosed by the present invention, download the genus of gibbon from the NCBI public database. The COI gene sequence corresponding to the white-handed gibbon (Hylobates lar, GenBank accession number. NC_002082).

The COI gene sequence corresponding to the white-handed gibbon is directly aligned with Table 5. For example, the variation sites of nucleotides 10, 42, 60, 61, and 66 in the white-handed gibbon are GTACT, which is neither ATGTC (Eastern White-browed Gibbon) nor GCACY (Northern White-cheeked Gibbon), indicating that the source of the sample is neither Eastern White-cheeked Gibbon The white-browed gibbon is also not the northern white-cheeked gibbon.

sequence listing

<110> Southwest Forestry University

<120> Molecular biological method for rapid identification of Eastern white-browed gibbon and northern white-cheeked gibbon

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<212> DNA

<213> The complete sequence DNA of the CO1 gene of the eastern white-browed gibbon "Beibei"

<400> 1

atgttcgccaaccgctggttattctccacaaaccataaagatattggaacactatacttgttattcggcgcatgagccggagtcctgggcacggccctaagcctccttatccgagccgaactgggtcaacccggtaatcttctgggcaatgaccacatctataacgtcatcgtaacagcccacgcattcgtcataatctttttcatagttatacctatcataattggaggctttggcaattgactagtccctttaataatcggcgcccccgacatggcattcccccgtataaataacataagcttctgactccttcctccctccttcctattactgcttgcatctgccatagtagaagccggcgccggaacgggatgaacggtctaccccccgctagcaggaaattattcccacccaggagcctctgttgacctaaccattttttctctacacttggccggagtatcgtccatcctaggagctatcaacttcattaccacaatcatcaacataaaacccccagctatatctcaatatcaaacacccctctttgtctgatccgtcctaatcacagcagtcctactccttctctccctaccagtcttagccgccggcattaccatgctactaacggaccgcaatctcaacaccactttctttgacccagctggaggaggagaccctattctataccaacacctattctgattcttcggccaccccgaagtttacattctaatcctaccaggctttggaataatttcacacatcgtaacacactactcaggaaaaaaagagccgttcggatatataggcatagtctgagccataatatcaattggcttcctaggtttcattgtctgagcccaccatatattcacggtaggcatagacgtagacacacgagcctattttacttccgctaccataattattgccatccccaccggtgttaaagtatttagctggctcgccacactccacggaagtgacaccaaat ggtccgccgcagtgctctgagccctaggattcattttcctctttacagtaggaggcctaaccggcatcgtactagcaaattcatcactggatattgtactccatgacacatactatgtcgtggcccatttccactacgtcctatccataggagccgtattcgccatcataggaggcttcgtccactgattccccctattctcgggctacactttagaccaaacctacgccaaaattcactttgccattatatttgtaggggtaaatttaaccttcttcccacaacacttcctcggcctgtccggaatgccacgacgttactctgattaccttgatgcgtatactacctgaaacatcctatcctctgtaggctcatttatctccctaacagcagtaatactaataatcttcataatctgagaggctttcgcttataagcgaaaaatcctaataatcgaacaaccttccactaacctagagtggttgtacggatgcccgccaccttatcacacatttgaagaacccgtctatataaaacctaga

<210> 1

<211> 1542

<212> DNA

<213> The complete sequence DNA of the CO1 gene of the eastern white-browed gibbon "Guagua"

<400> 2

atgttcgccaaccgctggttattctccacaaaccataaagatattggaacactatacttgttattcggcgcatgagccggagtcctgggcacggccctaagcctccttatccgagccgaactgggtcaacctggtaatcttctgggcaatgaccacatctataacgtcatcgtaacagcccacgcattcgtcataatctttttcatagttatacctatcataattggaggctttggcaattgactagtccctttaataatcggcgcccccgacatggcattcccccgtataaataacataagcttctgactccttcctccctccttcctattactgcttgcatctgccatagtagaagccggcgccggaacgggatgaacggtctaccccccgctagcaggaaattattcccacccaggagcctccgttgacctaaccattttttctctacacttggccggagtatcgtccatcctaggagctatcaacttcattaccacaatcatcaacataaaacccccagctatatctcaatatcaaacacccctctttgtctgatccgtcctaatcacagcagtcctactccttctctccctaccagtcttagccgccggcattaccatgctactaacggaccgcaatctcaacaccactttctttgacccagctggaggaggagaccctattctataccaacacctattctgattcttcggccaccccgaagtttacattctaatcctaccaggctttggaataatttcacacatcgtaacacactactcaggaaaaaaagagccattcggatatataggcatagtctgagccataatatcaattggcttcctaggtttcattgtctgagcccaccatatattcacggtaggcatagacgtagacacacgagcctattttacttccgctaccataattattgccatccccaccggtgttaaagtatttagctggctcgccacactccacggaagtgacaccaaat ggtccgccgcagtgctctgagccctaggattcattttcctctttacagtaggaggcctaaccggcatcgtactagcaaattcatcactggatattgtactccatgacacatactatgtcgtggcccatttccactacgtcctatccataggagccgtattcgccatcataggaggcttcgtccactgattccccctattctcgggctacactttagaccaaacctacgccaaaattcactttgccattatatttgtaggggtaaatttaaccttcttcccacagcacttcctcggcctgtccggaatgccacgacgttactctgattaccttgatgcgtatactacctgaaacatcctatcctctgtaggctcatttatctccctaacagcagtaatactaataatcttcataatctgagaggctttcgcttataagcgaaaaatcctaataatcgaacaaccttccactaacctagagtggttgtacggatgcccgccaccttatcacacatttgaagaacccgtctatataaaacctaga

<210> 1

<211> 1542

<212> DNA

<213> Northern white-cheeked gibbon "Lele" CO1 gene complete sequence DNA

<400> 3

atgttcgccgaccgctggttattctccacaaaccataaagacattggaacactatacttactatttggtgcatgagctggagtcctaggcacagctctaagcctcctcatccgagccgaactgggtcagcctggcaacctcctgggcaacgaccatatttataatgtcatcgtgacagcccacgcattcgtcataatctttttcatagtaatacctatcatgattggaggctttggtaactgactagttcctctaataatcggtgcccccgacatggcattcccccgtatgaataacataagcttctgactcctccctccctctttcctactactacttgcatccgccatagtagaagccggcgccggaacaggatgaacagtataccccccactagcaggaaactactcccacccaggagcctccgttgacctaaccatcttttccctccacctggccggagtatcatccatcctaggagctatcaattttattactacaatcatcaacatgaaacccccagccatgtctcaatatcaaacacccctctttgtctgatccgtcctaattacagcagtcctactccttctctctttaccagttctagccgccggcattaccatactattaacggatcgcaacctcaacaccactttctttgacccagccggaggaggagaccccattctatatcaacacctattctgattcttcggccaccccgaggtttatattctcattctgccaggcttcggaataatctcacacatcgtgacacactactcaggaaaaaaggagccatttgggtatatgggcatagtctgagccataatatcaattggcttcctaggctttattgtctgagcccaccatatattcacagtaggcatagacgtagacacacgagcctacttcacctccgctaccataattattgccattcccactggcgtcaaagtatttagctgactcgccacactccacggaagcgacaccaaat gatccgctgcagtactctgagccctaggcttcatcttcctctttacagtaggaggcttaactggcatcgtattagcaaactcatcactagacattgtacttcatgacacgtactatgttgtagcccacttccactacgtcttatccatgggggccgtattcgccatcatgggaggcttcgtccactgatttcccctgttctcaggctacactttagaccaaacctatgccaaaattcactttgctattatatttgtaggagtaaatttaaccttctttccacagcacttccttggcctgtccggaataccacgacgttactccgactaccccgacgcatacaccacctgaaacatcctatcctctgtaggctcgttcatctccctaacagcagtaatactaataattttcatggtctgagaggctttcgcctcaaaacgaaaaattctaataatcgaacagccttccactaacctagagtgattgtacggatgcccaccaccctatcacacatttgaagagcccgtctatataaaacctaga

<210> 1

<211> 1542

<212> DNA

<213> Northern white-cheeked gibbon "Wei" CO1 gene complete sequence DNA

<400> 4

atgttcgccgaccgctggttattctccacaaaccataaagacattggaacactatacttactatttggtgcatgagctggagtcctaggcacagctctaagcctcctcatccgagccgaactgggtcagcctggcaacctcctgggcaacgaccatatttataatgtcatcgtgacagcccacgcattcgtcataatctttttcatagtaatacctatcatgattggaggctttggtaactgactagttcctctaataatcggtgcccccgacatggcattcccccgtatgaataacataagcttctgactcctccctccctctttcctactactacttgcatccgccatagtagaagccggcgccggaacaggatgaacagtataccccccactagcaggaaactactcccacccaggagcctccgttgacctaaccatcttttccctccacctggccggagtatcatccatcctaggagctatcaattttattactacaatcatcaacatgaaacccccagccatgtctcaatatcaaacacccctctttgtctgatccgtcctaattacagcagtcctactccttctctctttaccagttctagccgccggcattaccatactattaacggatcgcaacctcaacaccactttctttgacccagccggaggaggagaccccattctatatcaacacctattctgattcttcggccaccccgaagtttatattctcattctgccaggcttcggaataatctcacacatcgtgacacactactcaggaaaaaaggagccatttgggtatatgggcatagtctgagccataatatcaattggcttcctaggctttattgtctgagcccaccatatattcacagtaggcatagacgtagacacacgagcctacttcacctccgctaccataattattgccatccccactggcgtcaaagtatttagctgactcgccacactccacggaagcgacaccaaat gatccgctgcagtactctgagccctaggcttcatcttcctctttacagtaggaggcttaactggcatcgtattagcaaactcatcactagacattgtacttcatgacacgtactatgttgtagcccacttccactacgtcttatccatgggggccgtattcgccatcatgggaggcttcgtccactgatttcccctgttctcaggctacactttagaccaaacctatgccaaaattcactttgctattatatttgtaggagtaaatttaaccttctttccacagcacttccttggcctgtccggaataccacgacgttactccgactaccccgacgcatacaccacctgaaacatcctatcctctgtaggctcgttcatctccctaacagcagtaatactaataattttcatggtctgagaggctttcgcctcaaaacgaaaaattctaataatcgaacagccttccactaacctagagtgattgtacggatgcccaccaccctatcacacatttgaagagcccgtctatataaaacctaga

<210> 1

<211> 1551

<212> DNA

<213> The complete mitochondrial genome sequence of northern white-cheeked gibbon (NC_021957) downloaded from Genbank corresponds to the complete DNA sequence of COI gene

<400> 5

atgttcgccgaccgctggttattctccacaaaccataaagacattggaacactatacttactattcggtgcatgagctggagtcctaggcacagccctaagcctcctcatccgagccgaactgggtcagcctggcaacctcctgggcaacgaccatatttataatgtcatcgtgacagcccacgcattcgtcataatctttttcatagtaatacctatcatgattggaggctttggtaactgactagttcctctaataatcggtgcccccgacatggcattcccccgtatgaataacataagcttctgactcctccctccctctttcctactactacttgcatccgccatagtagaagccggcgccggaacaggatgaacagtataccccccactagcaggaaactactcccacccaggagcctccgttgacctaaccatcttttccctccacctggccggagtatcatccatcctaggagctatcaattttattactacaatcatcaacatgaaacccccagccatgtctcaatatcaaacacccctctttgtctgatccgtcctaattacagcagttctactccttctctctttaccagttctagccgccggcattaccatactattaacggatcgcaacctcaacaccactttctttgacccagccggaggaggagaccccattctatatcaacacctattctgattcttcggccaccccgaggtttatattctcattctgccaggcttcggaataatctcacacatcgtgacacactactcaggaaaaaaggagccatttgggtatatgggcatagtctgagccataatatcaattggcttcctaggctttattgtctgagcccaccatatattcacagtaggcatagacgtagacacacgagcctacttcacctccgctaccataattattgccattcccactggcgtcaaagtatttagctgactcgccacactccacggaagcgacaccaaat gatccgctgcagtactctgagccctaggcttcatcttcctctttacagtaggaggcttaactggcatcgtattagcaaactcatcactagacattgtacttcatgacacgtactatgttgtagcccacttccattacgtcttatccatgggggccgtattcgccatcataggaggcttcgtccactgatttcccctgttctcaggctacactttagaccaaacctatgccaaaattcactttgctattatatttgtaggagtaaatttaaccttctttccacagcacttccttggcctatccggaataccacgacgttactccgactaccccgacgcatacactacctgaaacatcctatcctctgtaggctcgttcatctccctaacagcagtaatactaataattttcatggtctgagaggctttcgcctcaaaacgaaaaattctaataatcgaacagccttccactaacctagagtgattgtacggatgcccaccaccctatcacacatttgaagagcccgtctatataaaacctaaacaaaaaagg

<400> 1

<210> 2

<211> 16

<212> DNA

<213> P1 forward primer

<400> 6

aaccgaacgc aaatca

<210> 3

<211> 16

<212> DNA

<213> P1 reverse primer

<400> 7

tctcgggcta cacttt

<210> 4

<211> 17

<212> DNA

<213> P2 forward primer

<400> 8

tccgctggca ggaaact

<210> 5

<211> 17

<212> DNA

<213> P2 reverse primer

<400> 9

ctttccacga ccacgcc

sequence list

Claims (3)

1. A molecular biological method for rapidly identifying a great brow ape and a great white cheek ape, comprising the following sequential steps:

(1) separating and extracting genome DNA from a to-be-detected object;

(2) carrying out PCR amplification on the COI gene complete sequence of the object to be detected by utilizing two groups of PCR primers:

the primer sequences of the two groups of PCR primers are respectively as follows:

P1F：5'-AACCGAACGCAAATCA-3'，

P1R：5'-AAAGTGTAGCCCGAGA-3'；

P2F：5'-TCCGCTGGCAGGAAACT-3'，

P2R：5'-GGCGTGGTCGTGGAAAG-3'；

carrying out PCR by taking the genomic DNA obtained in the step (1) as a template and the primers P1F and P1R to obtain a fragment APCR product; carrying out PCR by taking the P2F and the P2R as a primer pair to obtain a fragment B PCR product;

(3) after the well amplified fragment A PCR product and the fragment B PCR product are sent to a biological company for sequencing, splicing the fragment A and the fragment B, and cutting off redundant bases before and after the COI gene complete sequence to obtain the COI gene complete sequence of the object to be detected;

(4) species identification was performed according to the nucleotide sequence characteristic variation sites listed in the following table:

nucleotide sequence characteristic variation site of great brow ape and great white cheek ape

The sequence numbers of the sites in the table are based on the position of the complete sequence of the COI gene; the letter "M" in the table is a abbreviation of the great brow ape; letter "J" is shorthand for northern white buccal gibbon; the relevant base codes are R ═ A/G and Y ═ C/T.

2. The rapid identification method according to claim 1, wherein the reaction system of the PCR in step (2) is 50. mu.l, wherein the template DNA is 25ng, 1 × PCR buffer, 2.5mM Mgcl₂1mM dNTP, 2 mug/mul BSA, 2pM of forward primer and reverse primer respectively, and 1 unit of TaqDNA polymerase; the specific operation is that deionized water is added to adjust the volume to 50 mu l, and the paraffin oil is used for sealing the system; the PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min, and extension at 72 ℃ for 1 min; 35 are provided withAfter cycling, the extension was carried out for a further 10 minutes at 72 ℃.

3. The rapid authentication method according to claim 1, wherein: the object to be detected comprises venous blood, excrement, hair or urine samples of animals.

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